The overall objective of this application is to elucidate mechanisms by which anthrax lethal toxin contributes to the disease process. Lethal toxin is an essential virulence factor that allows Bacillus anthracis to colonize and kill the host. Furthermore, damage caused by lethal toxin is likely a contributing factor to the long-term pathologies displayed by surviving patients. It is critical, therefore, to gain a better understanding of how the toxin interacts with the host. Lethal factor is the enzymatic component of the toxin that cleaves mitogen activated protein kinase kinases after being delivered to the mammalian cell cytosol by the second toxin component, protective antigen. How lethal factor's enzymatic activity causes the cellular effects associated with the toxin is poorly understood. The first aim of this proposal is to determine how lethal toxin interferes with cvtokine expression. We have discovered that lethal toxin destabilizes interleukin-8 mRNA in primary human endothelial cells. It has been shown that many transcripts are post-transcriptionally regulated by specific elements in their 3'untranslated regions and by the proteins that bind these regions. We will identify regulatory proteins and mRNA elements that are targeted by lethal toxin action;and identify other transcripts that are destabilized by the toxin. Our second aim is to determine how and why lethal toxin induces caspase-independent programmed cell death in human macrophages, but not in the immature monocvtes from which they were derived. We will use a combination of cellular fractionation and immunofluorescence techniques to identify proteins that mediate cell death;and compare toxin-sensitive and resistant cells to determine cellular properties that confer susceptibility to lethal toxin. Our final aim is to isolate lethal factor mutants that can cleave some of its substrates, but not others, so that the contributions of individual substrates to pathogenic processes can be assessed. To accomplish this, we will screen random lethal factor mutants for ones that are able to kill some cell types, but not others. This work will potentially identify new substrate binding determinants that could be targeted by therapeutics. Relevance: The goal of our research is to understand how anthrax lethal toxin damages human cells. A better understanding of how the toxin works will help doctors treat patients with anthrax and help researchers discover new drug treatments.